Most living organisms are under constant assault from external harmful environmental factors such as pathogens and parasites, but can defend themselves against the harmful environmental factors due to their immune systems. Immune systems are divided into innate and adaptive immune systems according to how to recognize an external foreign substance. In contrast to invertebrates that have only an innate immune system, vertebrates, such as humans, have both an innate immune system and an adaptive immune system. An adaptive immune system, observed only in vertebrates, is a memory-dependent immune system that induces a continued immune response by recognizing respective structures of harmful foreign substances (so-called antigens) that have invaded the body and selectively creating antibodies specific to the antigens. On the other hand, an innate immune system, observed in both of vertebrates and invertebrates, is a memory-independent immune system that recognizes and quickly responds to a conserved element (i.e., a pattern) shared among pathogens. Up until several years ago, the innate immune system was recognized as a less specific, less developed defense mechanism than the adaptive immune system, which primarily protects the body from an invading foreign substance until the latter generates antibodies.
However, active research into the immune system at the molecular level has been performed in recent years, and it has been determined that the innate immune system plays a critical role in the activation of the adaptive immune system [Carroll, M. C. et al, Curr. Opin. Immunol. 10, 36-48 (1998); Ruslan, M. et al., Cell 91, 295-298 (1997)], bringing the importance of the innate immune system into prominence.
These facts suggest that the regulation of the innate immune system could lead to a change in the adaptive immune system. Thus, new conceptual approaches to the innate immune system are required in terms of the treatment of diseases and the development of new drugs. Recently, the innate immune system has been actively studied at the molecular level by many domestic and foreign researchers [Medzhitov, R. et al., Nature 388, 394-397 (1997)].
Research into the innate immune system has been conducted mainly using invertebrates having an innate immune system. In particular, insects have been used in many researches on the innate immune system. Recent research results at the to molecular level show that there are similarities between the innate immune system in insects and that in humans, and thus, research into the innate immune system has been actively done using various insects. [Medzhitov, R. et al., (1997) Nature 388, 394-397 (1997); Hultmark, D. (1994), Nature 367, 116-117 (1994); Wasserman, S. A. Mol. Biol. Cell. 4, 767-771 (1993)].
The immune system of insects can be divided into a cellular immune response and a humoral immune response. The humoral immune response includes the secretion of antibacterial proteins into the body fluid against the invasion of foreign substances, the induction of lectin recognizing a specific sugar in invaded foreign substances, the activation of pro-phenoloxidase (hereinafter, referred to as “pro-PO”) known to be associated with melanin production and others.
According to research results on the pro-PO activation system in insects, it has been found that a serine proteinase inhibitor selectively inhibits the activity of phenoloxidase (PO) [Ashida, M. et al., Biochem. Biophys. Res. Commun. 113, 562-568 (1983)]. It has been reported that pro-PO is activated by a pro-PO cascade mediated by pro-PO activating factors (PPAFs) having a serine proteinase property [Aspan, A et al., Insect Biochem., 21, 363-373 (1991)]. It has also been reported that the pro-PO cascade mediated by PPAFs is initiated by a so-called pattern such as beta-1,3-glucan which is a fungal cell wall constituent [Kwon, T. H. et al., Mol. Cells. 7, 90-97 (1997); Saul, S. et al., Archs. Insect Biochem. Physiol. 7, 91-103 (1988); Ashida, M., Bombyxi mori, Insect Biochem. 11, 57-65 (1981)], or lipopolysaccharide (LPS) and peptidoglycan (PGN) which are bacterial cell wall constituents [Saul, S. et al., Archs. Insect Biochem. Physiol. 7, 91-103 (1988); Ashida, M., Bombyxi mori, Insect Biochem. 11, 57-65 (1981); Pye, A. E., Nature 251, 610-613 (1974)].
Generally, PO exists as inactive proenzyme (zymogen). When activated, PO catalyzes the oxidation of diphenols to quinones to thereby produce melanin. It is known that PO contains a copper in its molecule. In particular, it is thought that PO of insects plays a critical role in defense mechanisms such as browning and sclerotization of insect cuticles, leakage of body fluid from a wound site for wound healing, and protection of the body against invasive pathogens.
Pro-PO associated with defense mechanisms of insects had been actively studied by many researchers for the past several decades, but had not been isolated, purified, or sequenced until pro-PO from the larvae of three insects, Drosophila melanogaster, Bombyx mori, and tobacco hornworm (Manduca sexta) was isolated and purified, and its amino acid sequence was determined in 1995 [Saul S. et al., Archs. Insect Biochem. Physiol., 5, 1-11 (1987); Fujimoto K. et al., PNAS, 92, 7769-7773; Kawabata T. et al., PNAS, 92, 7774-7778 (1995)].
Natural pattern-recognition proteins involved in the innate immune system of is insects have been identified, and their biological functions in the pro-PO cascade have been partially identified [Girardin S E, et al., J Biol. Chem. 278: 803283 (2003); Hugot J P et al., Curr Opin Immunol. 15: 593597 (2003)]. Furthermore, β-1,3-glucan-recognition proteins have been isolated and identified from the hemolymph of the larvae of Tenebrio molitor belonging to order Coleoptera, and the relationship between the β-1,3-glucan-recognition proteins and the pro-PO cascade has been reported. In addition, β-1,3-glucan-recognition proteins have been isolated and identified from the hemolymph of larvae of Holotrichia diomphalia (Korean black chafer), and the biological functions of the β-1,3-glucan-recognition proteins have been reported [Lee M H, et al., J Biol. Chem. 279(5):3218-27 (2000); Zhang R et al., J Biol. Chem. 278(43):42072-9. (2003)].
Recently, it has been reported that, among the identified pattern-recognition proteins, PGN-recognition proteins (PGRPs) isolated from the hemolymph of Holotrichia diomphalia larvae specifically recognize β-1,3-glucan, which is a fungal pattern, not PGN, and activate the pro-PO cascade [Lee M H, et al., J Biol. Chem. 279(5):3218-27 (2000)]. This shows that the relationship between the molecular mechanism of natural pattern-recognition proteins and the activation mechanism of the pro-PO cascade is yet to be identified.
It has been reported that the pro-PO cascade of the hemolymph of Tenebrio molitor larvae is activated by β-1,3-glucan, which is a fungal pattern. It has also been found that proteins specifically recognizing β-1,3-glucan are present in the hemolymph of Tenebrio molitor larvae. These results open new possibilities for the development of diagnostic reagents for detecting fungal infections [Zhang R et al., J Biol. Chem. 278(43):42072-9. (2003)].
A PO activation system, which includes a cascade pathway for PO activation, is easily triggered by internal factors activated in response to the invasion of pathogens or foreign substances or the degranulation of host blood cells, converting pro-PO to PO to thereby produce melanin from catecholamines. Thus, it is difficult to separate pattern-recognition proteins that specifically recognize patterns (such as PGN and (β-1,3-glucan) triggering the PO activation system.